In recent decades the blood supply has been periodically contaminated by emerging infectious microbes with sometimes devastating consequences. Pathogens, such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV), have caused many thousands of chronic infections and deaths in transfusion and transplant recipients prior to the implementation of post-tragedy screening. Although the current "hindsight" approach has resulted in a blood supply that is safe from known hazards, it is still vulnerable to unanticipated pathogens. We propose to validate a proactive approach to protect the blood supply against newly emerging agents and new strains of existing agents and, at the same time, enhance protection against agents that are already of concern. We will develop a high-throughput nucleic acid test for analysis on the Ibis T5000 mass spectroscopy platform;the single-tube assay will use inexpensive reagents and provide detection of the known blood-borne pathogens, new strains of these pathogens, and pathogens of concern that are not currently detected. The Ibis T5000 technology is uniquely suited to the fast, sensitive, and accurate detection and identification of known and newly emerging pathogens. This capability was successfully demonstrated during the Phase I portion of this work by the creation of a single assay that has the ability to readily detect members of the extended families of six pathogens. In the proposed Phase II study, we will complete the assembly of a comprehensive screening test that will cover a variety of pathogens. We will first develop and validate primer pairs for the detection of parasites associated with Chagas disease (Trypanosoma cruzi) and malaria (Plasmodium species, including falciparum, vivax, ovale and malariae). We will then validate the final single- tube multiplex blood screening test that will allow identification of all strains and variants of HIV-1 (Group M, subtypes A-G;Groups N and O), HIV-2, HTLV 1-3, HCV, HBV, all known flaviviruses (including the West Nile and Dengue viruses), all influenza viruses (including human and avian), parvoviruses (including B19), hepatitis A virus, human herpes viruses (1-3, 5,8, excluding EBV), trypanosomes, and Plasmodium species. Both calibrant and positive controls will be produced to support this assay. This screen will be fully validated and the system tested with large numbers of blinded samples and a large number of samples from blood bank specimens. The technology is extensible to a much broader range of pathogens and will eventually provide a "seamless net" to protect the blood supply from pathogens that cause infectious disease.